Evidence that bicarbonate is not the substrate in photosynthetic oxygen evolution

It is widely accepted that the oxygen produced by photosystem II of cyanobacteria, algae, and plants is derived from water. Earlier proposals that bicarbonate may serve as substrate or catalytic intermediate are almost forgotten, though not rigorously disproved. These latter proposals imply that CO2 is an intermediate product of oxygen production in addition to O2. In this work, we investigated this possible role of exchangeable HCO3- in oxygen evolution in two independent ways. (1) We studied a possible product inhibition of the electron transfer into the catalytic Mn4Ca complex during the oxygen-evolving reaction by greatly increasing the pressure of CO2. This was monitored by absorption transients in the near UV. We found that a 3,000-fold increase of the CO2 pressure over ambient conditions did not affect the UV transient, whereas the S(3) --> S(4) --> S(0) transition was half-inhibited by raising the O2 pressure only 10-fold over ambient, as previously established. (2) The flash-induced O2 and CO2 production by photosystem II was followed simultaneously with membrane inlet mass spectrometry under approximately 15% H2(18)O enrichment. Light flashes that revealed the known oscillatory O2 release failed to produce any oscillatory CO2 signal. Both types of results exclude that exchangeable bicarbonate is the substrate for (and CO2 an intermediate product of) oxygen evolution by photosynthesis. The possibility that a tightly bound carbonate or bicarbonate is a cofactor of photosynthetic water oxidation has remained.     

Photosynthetic water oxidation at high O2 backpressure monitored by delayed chlorophyll fluorescence

The atmospheric dioxygen is produced by photosynthetic organisms. This light-driven process culminates in what appears as one step: a four-electron abstraction from two water molecules bound to the Mn4Ca complex of photosystem II. Recently, an intermediate of the O2-producing reaction sequence was stabilized by elevated oxygen backpressure and detected by UV flash photometry [Clausen, J., and Junge, W. (2004) Nature 430, 480]. We scrutinized its properties by delayed chlorophyll fluorescence measurements. Half-suppression of oxygen evolution was observed at a similar O2 pressure of 2.3 bar, as previously, now with photosystem II membrane particles from spinach, without artificial electron acceptors, and at a high signal-to-noise ratio. The data are tentatively interpreted as the stabilization of a 2-fold oxidized state of the catalytic center (S2*) with bound peroxide and its slow conversion into the normal S2 state by the release of peroxide.     

IAA及KCN在黄瓜子叶离体叶绿体氧化还原作用过程中的相互作用

ＩＡＡ对于不同时期的叶绿体ＤＣＰＩＰ光还原均有促进效应。而对叶绿体的放氧效率的促进则随子叶叶岭的增长而下降至零。ＣＮ＾－（１０＾－３ｍｏｌ／Ｌ）存在时不同程度地抑制叶绿体的ＤＣＰＩＰ光还原和氧化放氧。而ＩＡＡ（１０＾－５,１０＾－４ｍｏｌ／Ｌ）则能消除ＣＮ＾－的抑制作用而仅表现ＬＡＡ原有的促进作用。同时,ＩＡＡ可以释放照光和不照光条件下ＫＣＮ对离体叶绿体分解外源Ｈ２Ｏ２的抑制。叶绿体光系统的光还原     

Singlet oxygen formation by a peroxidase, H2O2 and halide system.

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3-diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenylfuran and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme. Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.     

Glycolic Acid Labeling During Photosynthesis with CO(2) and Tritiated Water

Chlorella pyrenoidosa were allowed to photosynthesize for short periods of time in the presence of ¹⁴CO₂ and HTO. Analysis of tritium and ¹⁴C labeling of photosynthetic intermediate compounds showed that the T/¹⁴C ratio of glycolic acid was comparable to that of intermediate compounds of the photosynthetic carbon reduction cycle when photosynthesis was performed in nearly 100% oxygen and only slightly higher under steady-state conditions. It is concluded that formation of labeled glycolic acid as a consequence of its proposed hydrogen transport role in photosynthesis is quantitatively of limited importance compared to the net synthesis of glycolic acid from CO₂.     

Evaluation of the role of methional, 2-keto-4-methylthiobutyric acid and peroxidase in ethylene formation by Escherichia coli.

During growth of Escherichia coli strain SPA O in the presence of methionine, an intermediate accumulates in the medium. This intermediate reacts with 2,4-dinitrophenylhydrazine, and can be degraded to ethylene either enzymically or photochemically, the latter being stimulated by the addition of a flavin. The pH optimum for the photochemical degradation of this intermediate and 2-keto-4-methylthiobutyric acid (KMBA) is pH 3 whereas the optimum for methional is pH 6. The enzyme which converts the intermediate to ethylene also converts KMBA to ethylene and has many of the properties of a peroxidase including inhibition by catalase, cyanide, azide and anaerobiosis. The enzyme which synthesizes the intermediate is not known but requires oxygen and pyridoxal phosphate. A pathway for ethylene biosynthesis is proposed in which methionine is converted to KMBA which can be degraded either by peroxidase or in a flavin-mediated photochemical reaction. Its relevance to the properties of other ethylene-producing bacteria and to the proposed pathway of ethylene release by higher plants is discussed.     

Time-resolved infrared detection of the proton and protein dynamics during photosynthetic oxygen evolution

Photosynthetic oxygen evolution by plants and cyanobacteria is performed by water oxidation at the Mn(4)CaO(5) cluster in photosystem II. The reaction is known to proceed via a light-driven cycle of five intermediates called S(i) states (i = 0-4). However, the detailed reaction processes during the intermediate transitions remain unresolved. In this study, we have directly detected the proton and protein dynamics during the oxygen-evolving reactions using time-resolved infrared spectroscopy. The time courses of the absorption changes at 1400 and 2500 cm(-1), which represent the reactions and/or interaction changes of carboxylate groups and the changes in proton polarizability of strong hydrogen bonds, respectively, were monitored upon flash illumination. The results provided experimental evidence that during the S(3) → S(0) transition, drastic proton rearrangement, most likely reflecting the release of a proton from the catalytic site, takes place to form a transient state before the oxidation of the Mn(4)CaO(5) cluster that leads to O(2) formation. Early proton movement was also detected during the S(2) → S(3) transition. These observations reveal the common mechanism in which proton release facilitates the transfer of an electron from the Mn(4)CaO(5) cluster in the S(2) and S(3) states that already accumulate oxidizing equivalents. In addition, relatively slow rearrangement of carboxylate groups was detected in the S(0) → S(1) transition, which could contribute to the stabilization of the S(1) state. This study demonstrates that time-resolved infrared detection is a powerful method for elucidating the detailed molecular mechanism of photosynthetic oxygen evolution by pursuing the reactions of substrate and amino acid residues during the S-state transitions.     

温度变化对藻类光合电子传递与光合放氧关系的影响

由于直接测定藻类的光合速率耗时且不方便,研究者们常通过测定藻类光合电子传递速率的方式来间接反映其光合速率,理论上,以氧气产生来度量的总光合速率（PGross）与电子传递速率(ETR)之间应该存在很好的线性关系。然而,由于温度的变化会影响藻类的光呼吸等耗氧的生理过程从而影响光合作用中的氧气释放,因此温度可能会对PGross与ETR之间的线性关系产生影响。研究了温度变化对蛋白核小球藻（Cholorella pyrenoidosa）、菱形藻（Nitzschia sp.）和水生集胞藻（Synechocystis aquetilis Sauv.）的总光合放氧速率（PGross）与电子传递速率(ETR)之间比率的影响,结果表明PGross/ETR随温度的升高而降低,低温条件下PGross/ETR比值较高,说明在相同的电子传递速率的情况下水的光裂解产生的氧有更多的可以释放出来;在高温条件下PGross/ETR比值相对较低,说明高温条件下可能有相对更多的水光裂解产生的氧被用于耗氧的生理过程而没有释放出来。研究表明当温度发生变化时,光合放氧与电子传递之间并不呈线性关系,这说明将ETR作为实际光合生产的评价指标时要谨慎,不能不加分析地直接应用。     

Cooperative binding of oxygen to the water-splitting enzyme in the filamentous cyanobacterium Oscillatoria chalybea

In the filamentous cyanobacterium Oscillatoria chalybea photolysis of water does not take place in the complete absence of oxygen. A catalytic oxygen partial pressure of 15x10(-6) Torr has to be present for effective water splitting to occur. By means of mass spectrometry we measured the photosynthetic oxygen evolution in the presence of H(2)(18)O in dependence on the oxygen partial pressure of the atmosphere and analysed the liberations of (16)O(2), (16)O(18)O and (18)O(2) simultaneously. The observed dependences of the light-induced oxygen evolution on bound oxygen yield sigmoidal curves. Hill coefficient values of 3.0, 3.1 and 3.2, respectively, suggest that the binding is cooperative and that four molecules of oxygen have to be bound per chain to the oxygen evolving complex. Oxygen seems to prime the water-splitting reaction by redox steering of the S-state system, putting it in the dark into the condition from which water splitting can start. It appears that in O. chalybea an interaction of oxygen with S(0) and S(1) leads to S(2) and S(3), thus yielding the typical oxygen evolution pattern in which even after extensive dark adaptation substantial amounts of Y(1) and Y(2) are found. The interacting oxygen is apparently reduced to hydrogen peroxide. Mass spectrometry permits to distinguish this highly specific oxygen requirement from the interaction of bulk atmospheric oxygen with the oxygen evolving complex of the cyanobacterium. This interaction leads to the formation H(2)O(2) which is decomposed under O(2) evolution in the light. The dependence on oxygen-partial pressure and temperature is analysed. Structural peculiarities of the cyanobacterial reaction centre of photosystem II referring to the extrinsic peptides might play a role.     

Water oxidation by photosystem II: H(2)O-D(2)O exchange and the influence of pH support formation of an intermediate by removal of a proton before dioxygen creation

Understanding the chemistry of photosynthetic water oxidation requires deeper insight into the interrelation between electron transfer (ET) and proton relocations. In photosystem II membrane particles, the redox transitions of the water-oxidizing Mn complex were initiated by nanosecond laser flashes and monitored by absorption spectroscopy at 360 nm (A(360)). In the oxygen evolution transition (S(3) + hν → S(0) + O(2)), an exponential decrease in A(360) (τ(O(2)) = 1.6 ms) can be assigned to Mn reduction and O(2) formation. The corresponding rate-determining step is the ET from the Mn complex to a tyrosine radical (Y(Z)(ox)). We find that this A(360) decrease is preceded by a lag phase with a duration of 170 ± 40 μs (τ(lag) at pH 6.2), indicating formation of an intermediate before ET and O-O bond formation and corroborating results obtained by time-resolved X-ray spectroscopy. Whereas τ(O(2)) exhibits a minor kinetic isotope effect and negligible pH dependence, formation of the intermediate is slowed significantly both in D(2)O (τ(lag) increase of ～140% in D(2)O) and at low pH (τ(lag) of 30 ± 20 μs at pH 7.0 vs τ(lag) of 470 ± 80 μs at pH 5.5). These findings support the fact that in the oxygen evolution transition an intermediate is created by deprotonation and removal of a proton from the Mn complex, after Y(Z)(ox) formation but before the onset of electron transfer and O-O bond formation.     

Transient inhibition by ribose 5-phosphate of photosynthetic O2 evolution in a reconstituted chloroplast system.

Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO2 fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O2 evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected. The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient DL-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity. It is concluded that ribose 5-phosphate inhibits photosynthetic O2 evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.     

Coregulation of electron transport and Benson-Calvin cycle activity in isolated spinach chloroplasts: studies on glycerate 3-phosphate reduction

Glycerate 3-phosphate-dependent O2 evolution was measured in intact chloroplasts in the absence of CO2. At all concentrations of added glycerate 3-phosphate oxygen evolution ceased before stoichiometric amounts of oxygen were evolved. The inhibition of glycerate 3-phosphate-dependent-O2 evolution increased with increasing concentrations of substrate added. A similar response was observed in chloroplasts treated with KCN which inhibits ribulose-1,5-bisphosphate carboxylase-oxygenase. Oxygen uptake via the oxygenase activity of this enzyme is therefore not the cause of the discrepancy in stoichiometry of oxygen release in this system. The addition of NaHCO3 to chloroplasts in which oxygen evolution was inhibited by glycerate 3-phosphate caused an immediate sustained rate of oxygen evolution in the absence of KCN but not with KCN present. Simultaneous measurements of chlorophyll a fluorescence showed that qQ remained oxidized, although net O2 evolution had ceased. As O2 evolution decreased, qE and delta pH increased. Upon the addition of the NaHCO3, QA became more oxidized while delta pH and qE were decreased, suggesting that the inhibition of electron transport at high glycerate 3-phosphate concentrations was mediated by photosynthetic control via delta pH. However, the levels of ATP, ADP, ribulose 1,5-bisphosphate, and Pi concentrations and ATP/ADP ratio. The stromal glycerate 3-phosphate content declined upon illumination until O2 evolution ceased. At this time a constant stromal glycerate 3-phosphate concentration of 8-10 mM was maintained while net import of glycerate 3-phosphate into the stroma had virtually ceased. The stromal triosephosphate content remained at a constant low level throughout but the glycerate 3-phosphate level increased slightly after addition of NaHCO3. The data provided by the measurements of thylakoid reactions and stromal metabolites suggest that photosynthetic electron transport is tightly coupled to the requirements of the stroma for ATP and NADPH. Glycerate 3-phosphate reduction requires much less ATP than the operation of the complete Benson-Calvin cycle since the stoichiometry of ATP and NADPH utilization is reduced to 1:1. We conclude that thylakoid electron flow is not sufficiently flexible to maintain NADPH and ATP production in the ratio of 1:1. This situation will favor overenergization of the thylakoid membrane, increased leakiness of protons, increased electron drainage to O2, and result in progressive inhibition of noncyclic electron flow.     

H2O2-Induced Inhibition of Photosynthetic O2 Evolution by Anabaena variabilis Cells

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN-, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ~20% of the total manganese pool. This content remained unchanged upon the addition of CN- and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophyll a and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.     

Enhancement of photosynthetic O2 evolution in Chlorella vulgaris under high light and increased CO2 concentration as a sign of acclimation to phosphate deficiency.

The photosynthetic oxygen evolution of Chlorella vulgaris (Beijer.) cells taken from phosphate-deficient (-P) and control cultures was measured during 8 days of culture growth. Under inorganic carbon concentration (50 microM) in the measuring cell suspension and irradiance (150 micromol m(-2) s(-1)), the same as during culture growth, there were no marked differences in the photosynthetic O2 evolution rate between the -P cells and the controls. The much slower growth of -P cultures indicated that the utilization of absorbed photosynthetically active radiation (PAR) in the CO2 assimilation and biomass production were in -P cells less efficient than in the controls. Alga cells under the phosphorus stress utilized more of the absorbed PAR in the nitrate reduction than the control cells. However, under conditions of more efficient CO2 supply (inorganic carbon concentration 150 microM, introducing of exogenous carbonic anhydrase to the measuring cell suspension) and under increased irradiance (500 micromol m(-2) s(-1)), the photosynthetic O2 evolution in -P cells reached a higher rate than in the controls. The results suggest that in -P cells the restricted CO2 availability limits the total photosynthetic process. But under conditions more favorable for the CO2 uptake and under high irradiance, the -P cells may reveal a higher photosynthetic oxygen evolution rate than the controls. It is concluded that an increased potential activity of the photosynthetic light energy absorption and conversion in the C. vulgaris cells from -P cultures is a sign of acclimation to phosphorus stress by a sun-type like adaptation response of the photosynthetic apparatus.     

Evidence for the occurrence of photorespiration in synurophyte algae

The fluxes of CO(2) and oxygen during photosynthesis by cell suspensions of Tessellaria volvocina and Mallomonas papillosa were monitored mass spectrometrically. There was no rapid uptake of CO(2,) only a slow drawdown to compensation concentrations of 26 μM for T. volvocina and 18 μM for M. papillosa, when O(2) evolution ceased, indicating a lack of active bicarbonate uptake by the cells. Darkening of the cells after a period of photosynthesis did not cause rapid release of CO(2), indicating the absence of an intracellular inorganic carbon pool. However, upon darkening a brief burst of CO(2) was observed similar to the post-illumination burst characteristic of C(3) higher plants. Treatment of the cells of both species with the membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide had no adverse effect on photosynthetic rate, but stimulated the dark CO(2) burst indicating the dark oxidation of a compound formed in the light. In the absence of any active accumulation of inorganic carbon photosynthesis in these species should be inhibited by O(2). This was investigated in four synurophyte species T. volvocina, M. papillosa, Synura petersenii, and Synura uvella: photosynthetic O(2) evolution rates in all four algae, measured by O(2) electrode, were significantly higher (40-50%) in media at low O(2) (4%) than in air-equilibrated (21% O(2)) media, indicating an O(2) inhibition of photosynthesis (Warburg effect) and thus the occurrence of photorespiration in these species.     

Ion and oxygen fluxes in the unicellular alga Eremosphaera viridis

Plasma membrane fluxes of the large unicellular model algal cell Eremosphaera viridis (De Bary) were measured under various light regimes to explore the role of plasma membrane fluxes during photosynthesis and high light-induced chloroplast translocation. Plasma membrane fluxes were measured directly and non-invasively with self-referencing ion-selective (H(+), Ca(2+), K(+) and Cl(-)) potentiometric microelectrodes and oxygen amperometric microelectrodes. At light irradiances high enough to induce chloroplast migration from the cell periphery to its center, oxygen evolution declined to respiratory net O(2) uptake prior to any significant chloroplast translocation, while net K(+) and Cl(-) influx increased during the decline in photosynthetic activity (and the membrane potential depolarized). The results suggest that chloroplast translocation is not the cause of the cessation of O(2) evolution at high irradiance. Rather, the chloroplast translocation may play a protective role: shielding the centrally located nucleus from damaging light intensities. At both high and low light intensities (similar to ambient growth conditions), there was a strong inverse correlation between H(+) net fluxes and respiratory and photosynthetic net O(2) fluxes. A similar inverse relationship was also observed for Ca(2+) net fluxes, but only at higher light intensities. The net H(+) fluxes are small relative to the buffering capacity of the cell, but are clearly related to both photosynthetic and respiratory activity.     

The Relation between Photosynthesis, Respiration, and Crassulacean Acid Metabolism in Leaf Slices of Aloe arborescens Mill

Leaves and leaf slices from Aloe arborescens Mill. were used to study the interrelations between Crassulacean acid metabolism, photosynthesis, and respiration. Oxygen exchange of leaf slices was measured polarographically. It was found that the photosynthetic utilization of stored malic acid resulted in a net evolution of oxygen. This oxygen production, and the decrease in acid content of the leaf tissue, were completely inhibited by amytal, although the rate of respiratory oxygen uptake was hardly affected by the presence of this inhibitor of mitochondrial electron transport. Other poisons of respiration (cyanide) and of the tricarboxylic acid cycle (trifluoroacetate, 2-diethyl malonate) also were effective in preventing acid-dependent oxygen evolution. It is concluded that the mobilization of stored acids during light-dependent deacidification of the leaves depends on the operation of the tricarboxylic acid cycle and of the electron transport of the mitochondria.     

钝顶螺旋藻在不同光照条件下的放氧特性

钝顶螺旋藻在持续照光和中等频率 (0.01~20 Hz) 的光/暗交替照光下的放氧特性对光生物反应器的设计和操作具有重要意义。构建了一套可实现光/暗交替的光生物反应器系统对此进行研究,结果显示：根据与放氧速率的关系,可以将光强分为4个区：光限制区 (0~335 μmol/(m2·s)),过渡区 (335~875 μmol/(m2·s)),光饱和区 (875~2 775 μmol/(m2·s)) 以及光抑制区 (2 775 μmol/(m2·s)以上)。提高光/暗频率能否提高微藻光合速率取决于所采用的光强和     

Water oxidation chemistry of photosystem II

The O(2)-evolving complex of photosystem II catalyses the light-driven four-electron oxidation of water to dioxygen in photosynthesis. In this article, the steps leading to photosynthetic O(2) evolution are discussed. Emphasis is given to the proton-coupled electron-transfer steps involved in oxidation of the manganese cluster by oxidized tyrosine Z (Y(*)(Z)), the function of Ca(2+) and the mechanism by which water is activated for formation of an O-O bond. Based on a consideration of the biophysical studies of photosystem II and inorganic manganese model chemistry, a mechanism for photosynthetic O(2) evolution is presented in which the O-O bond-forming step occurs via nucleophilic attack on an electron-deficient Mn(V)=O species by a calcium-bound water molecule. The proposed mechanism includes specific roles for the tetranuclear manganese cluster, calcium, chloride, Y(Z) and His190 of the D1 polypeptide. Recent studies of the ion selectivity of the calcium site in the O(2)-evolving complex and of a functional inorganic manganese model system that test key aspects of this mechanism are also discussed.